Nobuaki Maeda

A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta.

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase ζ (PTPζ) with high affinity. In this study, we examined the binding of midkine to PTPζ by solid-phase binding assay. Midkine and pleiotrophin binding to PTPζ were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nm) and high (0.58 nm) affinity binding sites. These results suggested that PTPζ is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPζ binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPζ exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPζ by interacting with the chondroitin sulfate portion of this receptor.

Multiple receptor-like protein tyrosine phosphatases in the form of chondroitin sulfate proteoglycan

The possibility that some of the brain proteoglycans are receptor‐like protein tyrosine phosphatases (PTPases) was investigated. Membrane‐bound proteoglycan fractions were prepared from the postnuclear membrane fraction of 8‐day‐old rat brain by DEAE ion‐exchange chromatography and CsCl density gradient centrifugation. The isolated proteoglycan fractions showed high PTPase specific activities together with the typical PTPase characteristics. Renaturation experiments indicated that chondroitin sulfate proteoglycans with 380‐ and 170‐kDa core proteins carried the PTPase activity. The proteoglycan with 380‐kDa core protein was identified as RPTPβ/ζ bearing HNK‐1 carbohydrate.

Neurons as well as astrocytes express proteoglycan-type protein tyrosine phosphatase ζ/RPTPβ: analysis of mice in which the PTPζ/RPTPβ gene was replaced with the LacZ gene

PTPζ/RPTPβ is a receptor-like protein tyrosine phosphatase expressed as a chondroitin sulfate proteoglycan. We generated mice in which the PTPζ gene was replaced by the LacZ gene by gene targeting. Analysis of heterozygous PTPζ-targeted mice allowed us to identify PTPζ-producing cells during development by examining expression of the LacZ gene. LacZ expression was detected only in the central nervous system throughout development from embryonic day 8.5. In the postnatal period, subsets of neurons and astrocytes in the brain, including pyramidal cells and astrocytes in the hippocampus, expressed LacZ. Primary cultures of cells from the cerebral cortex of embryonic day 16 mice also indicated that both neurons and astrocytes were positive for LacZ. These results indicated that neurons and astrocytes express PTPζ.

Neuroglycan C, a Novel Membrane-spanning Chondroitin Sulfate Proteoglycan That Is Restricted to the Brain

Monoclonal antibodies were raised to membrane-bound proteoglycans derived from rat brain, and four monoclonal antibodies that recognized a 150-kDa chondroitin sulfate proteoglycan with a core glycoprotein of 120 kDa were obtained. Immunohistological study revealed that the proteoglycan was associated with developing neurons. We screened rat brain cDNA libraries using the four monoclonal antibodies and isolated overlapping cDNA clones that encoded the entire core protein of 514 amino acids plus a 30-residue signal peptide. The deduced amino acid sequence suggested an integral membrane protein divided into five structurally different domains: an N-terminal domain to which chondroitin sulfate chains might be attached, a basic amino acid cluster consisting of seven arginine and two lysine residues, a cysteine-containing domain, a membrane-spanning segment, and a C-terminal cytoplasmic domain of 95 amino acids. On Northern blots, the cDNA hybridized with a single mRNA of 3.1 kilobases that was detectable in brains of neonatal and adult rats but not in kidney, liver, lung, and muscle of either. The sequence of the proteoglycan did not exhibit significant homology to any other known protein, indicating that the proteoglycan, designated neuroglycan C, is a novel integral membrane proteoglycan.

Cell surface-associated extracellular distribution of a neural proteoglycan, 6B4 proteoglycan/phosphacan, in the olfactory epithelium, olfactory nerve, and cells migrating along the olfactory nerve in chick embryos.

The immunocytochemical and immuno-electron microscopic distribution of a neural proteoglycan (PG) was investigated with a monoclonal antibody, MAb 6B4, in the olfactory epithelium, the olfactory nerve, and the cells originating the epithelium and migrating along the olfactory nerve toward the forebrain in chick embryos. The PG recognized by MAb 6B4, that is 6B4 PG, in the brain of early postnatal rats, is identical to phosphacan. In chick embryos, immunoreactivity to 6B4 PG appeared on embryonic day (ED) 3-3.5 in a thin layer beneath the olfactory epithelium. It disappeared immediately, then becoming apparent in the bundles of the olfactory nerve. The immunoreactivity in the nerve bundles gradually increased during ED 5-11. On the other hand, cell surface-associated extracellular localization of the immunoreactivity was seen in the olfactory epithelium on ED 6 and afterwards. Immunofluorescent double-labeling of 6B4 PG and gonadotropin-releasing hormone (GnRH) revealed that the cell bodies of both GnRH-containing cells and other cells migrating along the olfactory nerve were surrounded by a rim immunoreactive to the PG. Under an electron microscope, the surfaces of the cell bodies and of the neurites in the nerve bundles were surrounded by deposits immunoreactive to 6B4 PG. These results indicate that 6B4 PG in chick embryos is one type of cell surface-associated extracellular matrix molecule, and that 6B4 PG covered the surfaces of migrating cells and of elongating olfactory nerve. The cell surface-associated extracellular localization of 6B4 PG found in the nasal region, taken together with the binding properties of this PG with cell adhesion molecules shown in rat brains, suggested that 6B4 PG played a role in guiding the migration of cells along the olfactory nerve in chick embryos.

Spatially and Temporally Regulated Modification of the Receptor-like Protein Tvrosine Phosphatase ζ/β isoforms with Keratan Sulphate in the Developing Chick Brain

Protein tyrosine phosphatase ζ (PTPζRPTPβ) is a proteoglycan‐type receptor‐like protein tyrosine phosphatase specifically expressed in the brain. In addition to the transmembrane form (PTPζ‐A), the extracellular splice variant (PTPζ‐S) occurs as a major soluble chondroitin sulphate proteoglycan in the brain. We prepared antibodies which specifically recognize PTPζ‐A and ‐S, and analysed the carbohydrate structures on the two PTPζ isoforms in the developing chick brain. lmmunoprecipitation experiments using these antibodies revealed that almost all of the keratan sulphate recognized by a monoclonal antibody (5D4) was exclusively bound to PTPζ‐A and PTPζ‐S. Addition of keratan sulphate to these proteoglycans markedly increased from embryonic day (E) 11, in contrast to the addition of LeX and HNK‐1 carbohydrates, which gradually increased during development in accordance with expression of the core proteins, suggesting that keratan sulphate modification plays some specific roles. Moreover, at the early embryonic stage keratan sulphate was observed only in several restricted regions, especially at boundary regions such as the roof plate of the tectum, the zona limitans intrathalamica in the diencephalon, and the mesencephalon‐metencephalon boundary. At the mesencephalon‐metencephalon boundary, keratan sulphate modification of PTPζ isoforms was specifically observed from E3 to E6 on a ring of cells encircling the neural tube and their radially oriented processes, which were identified as radial glial fibres. This expression pattern of keratan sulphate spatiotemporally corresponded well to the formation of the fovea isthmi, a groove separating the mesencephalon from the metencephalon. These results suggest that carbohydrates including keratan sulphate on PTPζ isoforms play important roles in brain development by modulating the cell‐cell and/or cell‐substrate interactions mediated by these molecules.